Acupuncture needle manipulation is associated with a biomechanical phenomenon, "needle grasp" resulting in increased resistance to needle pullout. Needle grasp is widely viewed as essential to the therapeutic effect of acupuncture, yet its mechanism is unknown. We have shown in a previous study in humans that needle grasp is markedly increased by manipulation of the acupuncture needle and involves connective tissue rather than muscle. In this study, we will test the working model that needle grasp involves mechanical signal transduction through connective tissue via: 1) winding of connective tissue around the needle, needle/tissue mechanical coupling, pulling of collagen fibers and local deformation of extracellular connective tissue matrix. We will perform quantitative histological measurements of human subcutaneous tissue biopsies after insertion and rotation of an acupuncture needle, together with measurement of the peak force necessary to pull the needle out of the tissue (pullout force) in anesthetized human subjects undergoing surgery. We will characterize the quantitative relationship between pull out force and the relative volume of collagen surrounding the needle after varying amounts of needle rotation performed with a computer-controlled acupuncture needling instrument (Aim 1). 2) Transduction of the mechanical signal created by matrix deformation into connective tissue fibroblasts with cytoskeletal actin polymerization. We will quantify the effect of needle rotation on connective tissue fibroblast actin polymerization in rat tissue explants using histochemistry and confocal microscopy. We will study temporal and spatial characteristics of these measurements using varying amounts of uni-directional and bi-directional needle rotation at acupuncture points and control points (Aim 2). 3) Activation of intracellular signaling pathways leading to changes in fibroblast gene expression. We will monitor gene expression, with and without acupuncture needle rotation, in mouse tissue explants. We will target genes coding for extracellular matrix components, growth factors and cytokines known to be released in response to mechanical stimuli, using comparative gene expression analysis (Aim3). We will test acupuncture points, control points, uni-directional and bi-directional needle rotation in all three aims. The result of these experiments will provide key, new insights into the nature of biomechanical, tissue, cellular and molecular responses to acupuncture needling. Understanding these mechanisms will permit improved clinical trials and expand clinical applications of acupuncture.